D-serine is an endogenous allosteric activator of the N-methyl-D-aspartate (NMDA1) receptor. In multiple clinical studies, D-serine administration has been shown to be effective at treating the positive, negative and cognitive deficits of schizophrenia. In order to observe clinical effects, however, D-serine had to be administered at high doses (2 g per day po) multiple times per day (TID or BID). One reason for the high and frequent dose is that D-serine undergoes oxidation by D-amino acid oxidase (DAAO), a flavoenzyme expressed in the liver, kidney, and brain. Only a fraction of the administered D-serine is thought to cross the blood brain barrier and act on the presumed target, the NMDA receptor. One additional issue with D-serine therapy is that the products of D-serine oxidation, hydroxy pyruvate and hydrogen peroxide, have been associated with nephrotoxicity.
In order to address these problems, co-administration of D-serine with a DAAO inhibitor has been suggested to lower the dose of D-serine required to treat schizophrenia symptoms and also to prevent unwanted side effects caused by the DAAO-catalyzed reaction. Early results using this approach have been promising: oral co-administration of D-serine with a prototype DAAO inhibitor, 5-chloro-benzo[d]isoxazol-3-ol (CBIO), significantly enhanced plasma and brain levels of D-serine in rats compared to D-serine alone. D. Ferraris, et al. J Med Chem 51 (2008) 3357-59. Further, co-administration of CBIO with D-serine normalized prepulse inhibition deficits in ddy mice similar to the normalization observed when using 10-fold higher doses of D-serine alone. K. Hashimoto, et al. Biol Psychiatry 65 (2009) 1103-6. Drug-like DAAO inhibitors with acceptable pharmacokinetics and toxicity profiles are being sought as a novel therapeutic for patients with schizophrenia. In the early preclinical characterization of these new drug candidates inhibitors are evaluated in rodents for their ability to increase plasma D-serine levels after oral co-administration. Plasma D-serine could also be a useful pharmacodynamic marker to establish dose and a biomarker of drug effect once DAAO inhibitors are in the clinic.
Currently there are two HPLC-based methods to measure D-serine in plasma. One involves D-serine extraction, derivatization using N-tert-butyloxycarbonyl-L-cysteine and o-phthaldialdehyde followed by HPLC separation with a C18 column and detection of fluorescent signal of derivatized D-serine. A. Hashimoto, et al. J Chromatogr 582 (1992) 41-48. The other method was originally implemented with rat brain microdialysis samples using two HPLC columns in tandem involving derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), separation of the derivatized amino acids in an ODS column followed by a chiral column separation and fluorimetric detection. T. Fukushima, et al. Biomed Chromatogr 18 (2004) 813-19. Both methods allow for the separation of D-serine from other amino acids present in plasma including L-serine. These methods require 40-70 min chromatographic separations and are not amenable to concomitant analysis of multiple samples. Consequently, analyses of D-serine time profiles in plasma after co-administration with DAAO inhibitors is time consuming.
There exists a need for a high-throughput (e.g., a 96-well-format) assay to monitor D-serine in plasma.